Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis.

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.

Temperature dependent autocleavage and applications of recombinant L-asparaginase from Thermococcus kodakarensis for acrylamide mitigation.

This manuscript describes enhancement of soluble production, auto-cleavage analysis and assessment of acrylamide mitigation potential of Tk2246, a plant-type L-asparaginase from Thermococcus kodakarensis. The gene encoding Tk2246 was cloned and expressed in Escherichia coli. Recombinant Tk2246 was produced mainly in insoluble form. Various strategies were utilized to enhance the soluble production, which significantly increased the soluble yield. Interestingly, recombinant Tk2246 was produced even without addition of the inducer, though relatively in a lower amount. To our surprise, Tk2246 was produced in partially cleaved form when the inducer was not added in the culture. When applied for acrylamide mitigation, Tk2246 reduced the acrylamide formation more than 80% in French fries, chapati and yeast-leavened bread. In addition to acrylamide mitigation, Tk2246 exhibited antistaling activity without loss of sensory properties of the food. High activity, thermostability and efficient acrylamide reduction capability make Tk2246 a potential candidate for industrial applications.

Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis.

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 µmol min-1 mg-1 towards l-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards l- or d-glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 µmol min-1 mg-1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol-1. To the best of our knowledge, this is the first characterization of a plant-type l-asparaginase from class Thermococci of phylum Euryarchaeota.

Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 µmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.

The erratic antibiotic susceptibility patterns of bacterial pathogens causing urinary tract infections.

Increasing trend of antibiotic resistance and expression of Extended Spectrum Beta Lactamases (ESBLs) are serious threats for public health as they render the treatment ineffective. Present study was designed to elucidate the antibiotic-susceptibility patterns of ESBL and non-ESBL producing E. coli and K. pneumoniae causing urinary tract infections so that the ineffective antibiotics could be removed from the line of treatment. The bacterial isolates obtained from the urine of patients visiting a tertiary health care facility were cultured for strain identification using API20E. Antimicrobial susceptibility and ESBL detection were done by Kirby-bauer diffusion technique. Almost 53.4 % isolates of E. coli and 24.5 % isolates of K. pneumoniae were found to be ESBL producers. The ESBL producing bacteria were found to be more resistant towards various antibiotics. The most effective drugs against E. coli ESBL isolates were imipenem (99.54 %), ampicillin-sulbactam (97.48 %), piperacillin-tazobactam (96.86 %), fosfomycin (94.51 %), amikacin (92.26 %) and nitrofurantoin (90.68 %). The most effective drugs against K. pneumoniae ESBL isolates were imipenem (97.62 %), piperacillin-tazobactam (95.35 %), ampicillin-sulbactam (90.48 %) and amikacin (88.37 %). The antibiotics having the highest resistance, particularly by the ESBL producers were amoxicillin clavulanic acid, sulphamethoxalzole/ trimethoprim, cefuroxime, cefpirome, ceftriaxone and ciprofloxacin. Most of the isolates showed multi drug resistance (MDR). High frequency of ESBL producing E. coli and K. pneumoniae were observed as compared to previous data. Penicillins, cephalosporins and some representatives of fluoroquinolones were least effective against the common UTIs and are recommended to be removed from the line of treatment.